Chromogenic substrates for detecting bacterial hydrolases

ABSTRACT

The invention concerns a chromogenous substrate for detecting the presence of at least one enzyme enzymatic activity. The invention also concerns a method for using such a substrate. The invention further concerns a method for identification based on such substrates and a device for implementing said identification method. The invention is characterized in that the substrate consists of at lease two molecules, a first molecule consisting of a non-chromogenous marker part associated with at least a specific target part for the enzyme and a second molecule consisting of a non-chromogenous part, and the non-chromogenous marker part, once it is released, reacts with the second molecule to form a chromogenous molecule. The invention is particularly applicable in bacteriology.

The present invention relates to the detection of enzymes of hydrolasetype, in particular peptidases, by using effective chromogenicsubstrates. The present invention also relates to a process and a devicefor identifying microorganisms which are both simple and reliable.

Specific substrates have been used for many years to determine thepresence or absence of enzymatic activities characteristic of bacteria.By the choice of substrates, depending on whether or not there is areaction, it is possible to characterize the nature of a genus ofbacteria or to differentiate between the species of a given bacterialgenus.

Synthetic substrates of enzymes consist of two portions, a first portionwhich is specific for the enzymatic activity to be detected, and asecond portion which acts as a label, and which is referred toherein-below as the labeling portion.

These specific substrates can be fluorescent or chromogenic substrates.In point of fact, it is the second portion or labeling portion which isfluorescent or chromogenic, when it is not combined with the firstportion.

Fluorescent substrates may be of diverse composition.

First of all, substrates based on umbelliferone or aminocoumarin, andthe derivatives thereof substituted in position 2, which allow therelease of a fluorescent compound whose color ranges from blue to greenunder an ultraviolet (UV) lamp (λ_(ex)=365 nm).

Next, substrates based on resorufin (and derivatives), in which there isrelease of a compound which is fluorescent pink under natural light(λ_(ex)=530 nm).

Finally, substrates based on fluorescein (and derivatives) which, afterdegradation, releases a compound which is fluorescent yellow undernatural light (λ_(ex)=485 nm).

These substrates are unsuitable for use in agar media, and are used morein liquid medium.

The chromogenic substrates may also be of diverse nature.

Firstly, there are substrates based on indoxyl and its derivativeswhich, in the presence of oxygen, produce a precipitate ranging fromblue to pink.

Their applications are essentially limited to osidases and esterases anddo not concern the detection of a peptidase activity. Whereas they arewell suited to use on a solid support (filter, agar, electrophoresisgel, etc.), they are less well suited to the use in liquid aqueousmedium (formation of a precipitate).

Secondly, there are substrates based on hydroxyquinoline or esculetinand their derivatives, which produce a brown precipitate in the presenceof iron salts.

In this case also, their applications are limited to osidases andesterases. They are suitable for use on a solid support, and relativelyunsuitable for use in liquid aqueous medium.

Thirdly, there are substrates based on nitrophenol and nitroaniline andderivatives, which lead to the formation of a yellow compound.

They make it possible to detect osidase and esterase activities in thecase of nitrophenol-based substrates, and peptidase activities in thecase of nitroaniline-based substrates. However, in the case of detectingpeptidase activities, the nitroaniline released is toxic to the bacteriawhich it is desired to identify or characterize, which may have anegative impact on current or subsequent analyses. Moreover, they arerelatively unsuitable for use on a solid support, and are better suitedto use in liquid medium. Furthermore, they are not particularlychromogenic on account of the relatively low extinction coefficient ofthe color (yellow) which gives a relatively weak contrast in biologicalmedia.

Fourthly, there are substrates based on naphthol and naphthylamine andits derivatives. In this case, the reaction is carried out in twostages; the naphthol or naphthylamine released by the enzymatic activityundergoes an “azo-coupling” in the presence of a diazonium salt which isadded at the detection stage, leading to the formation of a coloredinsoluble compound.

They make it possible to detect osidase and esterase activities by meansof naphthol, and peptidase activities by means of naphthylamine. Theazo-coupling reaction is carried out in a medium which is oftenchemically corrosive and toxic to bacteria, making the sample unusablefor other analyses, and, what is more, naphthylamines are carcinogenic.

To detect naphthylamine and thus a peptidase activity, it is alsopossible to add p-dimethylamino-cinnamaldehyde in acidic medium at theend of the enzymatic reaction, instead of a diazonium salt, althoughthis still has toxicity drawbacks with respect to the sample analyzed.

Patent application FR-A-2 708 286 proposes the use of a mixture ofchromogenic substrates, each chromogen giving a particular colorationfor a specific enzyme which is different from the coloration and enzymeassociated with the other chromogen. When the two colorations and thusthe two enzymes are present, there is formation of a “tertiarycoloration”.

However, this technique is unsatisfactory since, as a function of thelow concentration of one of the two enzymes, it is not possible todetect this enzymatic activity which is thus masked by the colorationassociated with the enzyme whose concentration is predominant.

Finally, patents U.S. Pat. No. 4,681,841 and U.S. Pat. No. 4,588,836describe an indirect method for detecting a single enzymatic activityusing a coupling between an aminobenzene and a hydroxyaromaticderivative (for example α-naphthol), this coupling leading to theformation of a chromogenic indicator in the presence of oxidase. One ofthe two compounds (the aminobenzene) forms part of the composition ofthe starting substrate; if the desired enzymatic activity is present,this compound will be released and will be able to react with the othercompound.

Nevertheless, in order for the detection of the desired enzyme to bepossible, the presence of oxidase in the reaction medium is an absolutenecessity. However, although this information discourages a personskilled in the art from looking for a solution which does not useoxidase, the Applicant has proved, by numerous tests carried out in itslaboratories, that it is possible to detect the desired enzyme by meansof using, for example, aminobenzene and α-naphthol, without the additionof oxidase.

It may thus be readily appreciated that no chromogenic substrate whichis particularly effective and advantageous as regards detecting at leastone peptidase activity currently exists.

As regards the identification process and device, the state of the artconsists of an identification process which involves a three-stepmanipulation:

taking a sample of the colony to be identified,

carrying out an orinetation test, and

looking for colonies similar to those observed and preparing aninoculum.

The orientation test should be performed before using an identificationsystem. This is especially the case for Gram staining which requires anobservation by microscope.

However, this coloration is not always easy to carry out and above allto interpret. Moreover, the cost of this test is far from negligible.

One of the aims of the present invention is thus to create a linkbetween a culture medium and antibiotic assay and identificationsystems, offering biologist the possibility of performing a simpleone-step test both for confirming the result of the Gram staining andfor preparing the inoculum. Thus, the choice of antibiotic assay andidentification tests is made reliable.

Patent application EP-A-0 122 028 proposes a colorimetric method fordetecting the presence of at least one enzyme suspected of being presentin a biological sample. It recommends the preparation of an absorbentbrush, absorbing in this material at least one susbstrate which isspecific for the enzyme which it is desired to detect. The absorbentmaterial containing the substrate(s) is dried before use.

The invention allows the colormetric detection of enzymatic activitiesof hydrolase (osidase, esterase, phosphatase or peptidase) type with theaid of synthetic substrates based on two compounds, as well as a novelprocess which can be applied to both liquid and solid reaction media.

The invention also proposes a process and a device for identifyingmicroorganisms, which orient the identification, and allow the recoveryof said microorganisms in order to allow an inoculum to be prepared.This inoculum makes it possible to use the same microorganisms for oneor more other steps, such as an identification or an antibiotic assay.

After hydrolyzing the substrates, the enzymatic activities are detectedon the basis of the formation of a colored complex from the oxidativecoupling of the two compounds mentioned above. This oxidative couplingcan be facilitated by an oxidizing agent added to the reaction medium orproduced during a metabolic process within this medium, or more simplyby the presence of endogenous oxygen in this same medium.

According to a first interpretation, the present invention relates to acombination of two distinct entities, one being a nonchromogenicmolecule and the other being the substrate for detecting the presence ofthe enzymatic activity or at least one enzyme. The substrate ischaracterized in that in consists of a specific portion of the enzyme,and of a nonchromogenic molecule other than the first molecule, whichconstitutes the labeling portion of the substrate; the twononchromogenic molecules react together when they are in free form andcan generate a chromogenic molecule.

Still according to this first interpretation, the invention also relatesto a combination of two distinct entities, each corresponding to adifferent substrate and making it possible to detect the presence of theenzymatic activity of at least two enzymes. Each substrate ischaracterized in that it consists of a specific portion of the desiredenzyme, and of a nonchromogenic molecule constituting the labelingportion of the substrate, and in that the two nonchromogenic moleculescorresponding to the two substrates react together when they are in freeform and can generate a chromogenic molecule.

According to a second interpretation, which will be used moreparticularly hereinbelow, to arrive at this result, it is necessary touse a new form of substrates. Thus, the substrate according to theinvention comprises two different molecules. One of the moleculesconsists of a specific portion, an enzymatic activity and a labelingportion, which is distinct for a chromogenic molecule. The othermolecule always comprises the labeling portion, distinct for achromogenic molecule, which is free or associated with at least onespecific portion, of an enzymatic activity. The object of the inventionis thus, if the enzymatic activity(ies) is (are) present, to reveal theformation of a chromogenic molecule which is effective only when the twolabeling portions, once released, have become associated.

To this end, the present invention relates to a chromogenic substratefor detecting the presence of the enzymatic activity of at least oneenzyme, characterized in that it consists of at least two molecules, afirst molecule consisting of a nonchromogenic labeling portionassociated with at least one specific target portion of the enzyme and asecond molecule consisting of another nonchromogenic substance, and inthat the nonchromogenic labeling portion, once released, reacts with thesecond molecule to form a chromogenic molecule.

The present invention also relates to a chromogenic substrate fordetecting the presence of the enzymatic activity of at least twoenzymes, characterized in that it consists of at least two molecules, afirst molecule consisting of a nonchromogenic labeling portionassociated with at least one specific target portion of the first enzymeand a second molecule consisting of another nonchromogenic labelingportion associated with a specific target portion of the second enzyme,and in that the nonchromogenic labeling portions, once released, reactto form a chromogenic molecule.

Irrespective of the method of preparation of the substrate, thenonchromogenic labeling portion of the first molecule consists ofaminobenzene or a derivative thereof:

that [sic] the nonchromogenic labeling portion of the second moleculeconsists of α-naphthol or a derivative thereof:

and that [sic] the chromogenic molecule obtained consists of:

In this case, the radical R₁ consists of —OH, —SH or

the radical R₁₀ consists of —H or an atom, such as —Br, —Cl or —I, or agroup of atoms, such as —SH, which can be removed during the oxidativecoupling, and each radical R₂ to R₉, R₁₁, or R₁₂ consists of —H, —OH,—Br, —Cl, —I or other more complex substituents, such as —CH₃, —CH₂CH₃,—OCH₃, —OCH₂CH₃ or —COOH.

In the case of the radical R₁, X/or Z consists of —H, or other morecomplex substituents, such as —CH₃, —CH₂CH₃,

According to one embodiment, at least one of the pairs of radicals R₂/R₃and R₄/R₅ consists of an aromatic, alicyclic or heterocyclic system.

The present invention also relates to a process for detecting anenzymatic activity via substrates, as described above, which consistsin:

placing the substrates in contact with bacteria of at least one type,

waiting for the bacteria to hydrolyze the substrates, and

detecting the enzymatic activities based on the formation of a coloredcomplex from the oxidative coupling of the two labeling portions.

Another object of the present invention is to propose a process, inwhich the molecules are present in an absorbent material and are placedin contact with the sample, and, after detection, the microorganismsthus withdrawn are resuspended in order to allow subsequent analyses(identifications, antibiotic assays, etc.).

The molecules and other compounds forming part of the reactioncomposition contained in the absorbent material are:

the first molecule consisting of aminobenzene or a derivative thereof,and

the second molecule consisting of α-naphthol or a derivative thereof.

The reaction composition also contains an oxidizing agent, such aspotassium ferricyanide.

Among the other compounds, it is also possible to have a reactionactivator, such as a small amount of the first molecule and/or of thesecond molecule, which is (are) present in the sample to be tested.

Still among the other compounds, it is possible for the composition tocomprise a binder or adhesive, such as PolyVinylPyrrolidone (PVP).

The process can be used for the Gram identification of a bacterialspecies to be tested and, in this case, the first molecule consists ofAlaDMpPD and that [sic] the second molecule consists of α-naphthol.

Furthermore, the composition of the reaction mixture absorbed by theabsorbent material is as follows:

α-naphthol from 0.01 g/l to 5 g/l, preferably from 0.1 g/l to 1 g/l, andfor example 0.5 g/l,

potassium ferricyanide from 0.01 g/l to 5 g/l, preferably from 0.1 g/lto 1 g/l, and for example 0.5 g/l, and

AlaDMpPd from 0.01 g/l to 5 g/l, preferably from 0.1 g/l to 1 g/l, andfor example 0.35 g/l.

According to one variant, the activator consists of Ala-DMpPD at aconcentration of from 0.01 g/l to 0.5 g/l, preferably from 0.05 g/l to0.1 g/l, and for example 0.075 g/l.

According to another variant, the composition also comprises from 1 g/lto 50 g/l, preferably from 10 g/l to 25 g/l, and for example 15 g/l, ofPVP.

The invention also relates to the use of substrates, as described above,for detecting an enzymatic activity of peptidase type.

Finally, the present invention relates to an identification device forcarrying out the identification process described above, which consistsof a support, for example made of plastic, which is inert with respectto the absorbent material and to the substrate(s) it contains and/orwith respect to the test sample, on which is mounted a head made of anabsorbent material, such as viscose.

I-Novel chromogenic substrates and implementation process:

The invention, which will be described below, relates to a few specificembodiments of the invention. These embodiments therefore do not limitthe scope of the present invention which can be used for detectingenzymatic activities of any type, of hydrolase type, and formicroorganisms of any genus or type.

The chromogenic substrate for detecting the presence of the enzymaticactivity of at least one enzyme consists of at least two molecules, afirst molecule consisting of a nonchromogenic labeling portionassociated with at least one specific target portion of the enzyme and asecond molecule consisting of another nonchromogenic substance. Thenonchromogenic labeling portion, once released, reacts with the secondmolecule to form a chromogenic molecule.

In another case, the chromogenic substrate for detecting the presence ofthe enzymatic activity of at least two enzymes consists of at least twomolecules, a first molecule consisting of a nonchromogenic labelingportion associated with at least one specific target portion of thefirst enzyme and a second molecule consisting of another nonchromogeniclabeling portion associated with a specific target portion of the secondenzyme. Once released, the nonchromogenic labeling portions react toform a chromogenic molecule.

In the case of the detection of only one enzymatic activity, there aretwo nonchromogenic molecules, i.e. a first molecule having a portionwhich is specific for the enzymatic activity to be detected and alabeling portion, whereas the second molecule consists only of thelabeling portion. The two labeling portions consist respectively ofcompounds I and II below:

in which:

R₁=—OH or —SH or

 with X and/or Z=—H or other more complex substituents, such as —CH₃,—CH₂CH₃,

R₁₀=—H or a group or atom which can be removed during the oxidativecoupling, such as —Br, —Cl, —I, —SH, etc.,

R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₁ and R₁₂=—H, —OH, —Br, —Cl, —I, —CH₃,—CH₂CH₃, —OCH₃, —OCH₂CH₃, —COOH, or any other more complex substituent,R₂/R₃ and R₄/R₅ also possibly forming part of an aromatic, alicyclic oreven heterocyclic system, such as:

Compound III obtained by combining the two compounds I and II is thus asfollows:

To understand the invention, it is recalled that the formula of ahydrolase enzyme substrate may be represented schematically in the form:

A—O—B, if it is an osidase, phosphatase, esterase or sulfatasesubstrate.

The enzymatic hydrolysis may then be written as:

A—O—B+H₂O→A—OH+B—OH

with, according to the invention:

A—OH=ose, phosphate, sulfate, fatty acid (from the simplest, i.e. aceticacid), and

B—OH=compound I with R₁=—OH or compound II.

A—CO—NH—B, if it is a peptidase substrate.

The enzymatic hydrolysis can then be written as:

A—CO—NH—B+H₂O→A—COOH+B—NH₂,

with, according to the invention:

A—COOH=amino acid or chain of amino acids possibly ending with ablocking agent, and

B—NH₂=compound I.

The process according to the invention uses, on the one hand, anenzymatic substrate as described above, based on:

either a compound I derived from aminobenzene, with R₁=—OH for theosidase, phosphatase or esterase substrates,

or a compound II derived from α-naphthol (with the exclusion ofpeptidase substrates), on the other hand, a revealing agent which isinitially present in the reaction medium or which can be added during orafter the enzymatic reaction, this agent being:

either compound II for the detection of compound I resulting from theenzymatic hydrolysis of the substrates based on the latter compound, and

or compound I for the detection of compound II resulting from theenzymatic hydrolysis of the substrates based on the latter compound.

The formation in the reaction medium of the colored compound III(derived from indophenol) from the oxidative coupling of I and II makesit possible to detect the hydrolysis of the substrate and thus theenzymatic activity involved.

The color of compound III depends on the substituents on compounds I andII.

For example, it is purple if R₁=—OH and R₂ to R₁₁=—H, or blue if R₁=—OH,R₂ and R₅=—Cl and R₃ to R₁₁=—H.

The process thus described is, in particular, extremely advantageous forcalorimetrically detecting activities of peptidase type. Specifically,according to the prior art, the choice of colored reactions was limitedto the use of substrates based on nitroaniline, which is relativelynonchromogenic, and of substrates based on naphthylamine, which ishighly toxic, necessitating the addition of a reagent at the end of thereaction (for example a diazonium salt) to detect the amine released.

The invention proposes a novel type of substrates for peptidases, basedon aminobenzene or derivatives (compound I).

Gradually as this compound I is released into the reaction medium duringthe enzymatic hydrolysis, it forms a strongly colored complex (generallycolored red to blue) by oxidative coupling with α-naphthol or aderivative (compound II) present in this medium.

The coupling reaction can also be obtained at the end of the test if theaddition of compound II is carried out at the end of the enzymaticreaction.

Another important advantage of the invention consists in combining twosubstrates in the reaction medium, according to the process, one basedon compound I and the other based on compound II.

Any type of combination of simultaneous detection of two enzymaticactivities (for example a peptidase and an osidase, an esterase and anosidase, two osidases, etc.) may thus be envisaged. In these cases, thecolored complex III will be formed only if the two substrates arehydrolyzed. This combination may be very useful for diagnostic testswhich require high specificity, in particular when it is a case ofcharacterizing microorganisms.

One of the examples mentioned illustrates the advantage of thissimultaneous detection of two enzymatic activities in the field ofidentifying bacteria.

Among the other advantages of the invention, it is possible to givecompound I a double functionality. Specifically, it is possible tochemically graft onto compound I, on the one hand onto the —NH₂ group,an amino acid or a chain of amino acids possibly ending with a blockingagent, and, on the other hand, onto R₁, if it is a hydroxyl (—OH) group,an ose, a phosphate or a fatty acid. A molecule is obtained which may betermed a double substrate and which, in order to generate compound I infree form in the reaction medium, should be hydrolyzed by two distinctenzymatic activities, on the one hand a peptidase, and on the other handan osidase, phosphatase or esterase, depending on the nature of theproduct grafted onto R₁.

Thus, in the presence of compound II, the colored complex III can onlybe obtained if the two enzymatic activities are present. The advantageof such substrates with double functionality lies in their very highspecificity which may be exploited to characterize microorganisms, forexample.

In the same sense, a mixture of a substrate with double functionalitybased on compound I, as described above, and a substrate based oncompound II may be envisaged according to the invention to search foranother osidase, phosphatase or esterase. In this case, the productionof the colored complex III requires the simultaneous presence of threeenzymatic activities, in order to release compounds I and II. Twoenzymatic activities make it possible to hydrolyze the molecule based oncompound I and one enzymatic activity makes it possible to hydrolyze themolecule based on compound II. The specificity of the test is thusfurther increased.

According to the invention, it is also possible to combine differenttypes of substrates in the same reaction medium, in particularsubstrates known in the prior art and substrates according to theprocess claimed. This makes it possible to carry out different coloredreactions, and thus to simultaneously detect several enzymaticactivities in the same reaction medium.

The invention can be applied to the search for enzymatic activities indifferent types of biological or nonbiological samples.

The invention can also be used to characterize microorganisms such asbacteria or yeasts. In this case, the enzymatic tests can be performedin liquid medium in individual tubes or in compartmentalized supports,such as microtitration plates, for example. They can also be performedin agar medium (for example in Petri dishes) with staining of thecolonies producing the desired enzymatic activities.

The present invention thus relates to a process for detecting anenzymatic activity by means of substrates, as described above, whichconsists in:

placing the substrates in contact with bacteria of at least one type,

waiting for the bacteria to hydrolyze the substrates, and

detecting the enzymatic activities based on the formation of a coloredcomplex from the oxidative coupling of the two labeling portions.

The oxidative coupling is facilitated either by:

the addition of at least one oxidizing agent to the reaction medium,

the production, during a metabolic process in this reaction medium, ofat least one oxidizing agent,

the presence of endogenous oxygen in said reaction medium.

The examples given below will allow the invention to be understood moreclearly. They relate specifically to the field of bacteriology, but itis clear that the process can be applied to any other field ofenzymology which involves looking for hydrolases.

EXAMPLE 1 Detection of β-glucosidase in Liquid Medium (method 1)

The reaction medium proposed in this example has the formulation:

meat peptone 10 g NaCl 5 g α-naphthyl-β-glucopyranoside 0.5 g4-aminophenol 50 mg H₂O 1000 ml pH 7.0

This medium is sterilized by filtration through a 0.22μ Millipore filterand distributed into sterile tubes at a rate of 5 ml per tube.

The test to search for β-glucosidase is performed on 4 strains culturedfor 24 h at 35-37° C. on Trypticase-Soya agar. These strains belong,respectively, to the species:

Escherichia coli,

Staphylococcus sciuri,

Streptococcus pyogenes,

Enterococcus faecalis.

Starting with the above cultures, aqueous suspensions adjusted to 0.5McFarland are prepared.

100 microliters of these suspensions are used to inoculate four tubes ofreaction medium, each tube corresponding to a strain.

The stoppered tubes are incubated at 35-37° C. for 18 to 24 h.

After incubation, the appearance of a purple coloration is observed,corresponding to the formation of the indophenol complex and reflectingthe presence of a β-glucosidase activity.

The results obtained are as follows:

Expected Strain theoretical result Result obtained E. coli − − S.sciuri + + S. pyogenes − − E. faecalis + +

EXAMPLE 2 Detection of β-glucosidase in Liquid Medium (method 2)

The reaction medium proposed in this example has the formulation:

meat peptone 10 g NaCl 5 g α-naphthol 50 mg4-aminophenol-β-D-glucopyranoside 0.5 g H₂O 1000 ml pH 7.0

The preparation of the medium, the choice of test strains and theimplementation of the test are identical to the procedure described inExample 1.

The appearance of a purple coloration due to the formation of theindophenol complex indicates the presence of a β-glucosidase activity.

The results obtained are identical to those of Example 1, i.e.:

Expected Strain theoretical result Result obtained E. coli − − S.sciuri + + S. pyogenes − − E. faecalis + +

These results show that it is possible to detect an activity of osidasetype using according to the invention either a substrate based oncompound I or a substrate based on compound II. This would likewise bethe case for the investigation of activities of esterase or phosphatasetype.

EXAMPLE 3 Detection of Pyroglutamyl-amino-peptidase in Liquid Medium:

The reaction medium proposed in this example has the formulation:

meat peptone 10 g NaCl 5 g α-naphthol 50 mg L-pyroglutamyl-4-amino- 0.5g 2,6-dichlorophenol H₂O 1000 ml pH 7.0

The preparation of the medium, the choice of test strains and theimplementation of the test are identical to the procedure described inExample 1.

The appearance of a blue coloration due to the formation of theindophenol complex reveals a pyroglutamyl-aminopeptidase activity. Itshould be noted that the blue color results from the presence ofchlorine groups in positions 2 and 6 on the 4-aminophenol.

The results obtained are as follows:

Expected Strain theoretical result Result obtained E. coli − − S. sciuri− − S. pyogenes + + E. faecalis + +

EXAMPLE 4 Combined Investigation of Two Enzymatic Activities Accordingto the Process of the Invention Application to the SimultaneousDetection of β-glucosidase and Pyroglutamyl-aminopeptidase

The reaction medium proposed in this example has the formulation:

meat peptone 10 g NaCl 5 g α-naphthyl-b-D-glucopyranoside 0.5 gL-pyroglutamyl-4-amino- 0.5 g 2,6-dichlorophenol H₂O 1000 ml pH 7.0

The preparation of the medium, the choice of test strains and theimplementation of the test are identical to the procedure described inExample 1.

The appearance of a blue coloration due to the formation of theindophenol complex is possible only if the two substrates arehydrolyzed. This therefore reflects a combination of β-glucosidase andpyroglutamyl-aminopeptidase activities in the same reaction medium. Theabsence of one of the activities prevents the formation of the coloredcomplex.

The results obtained are as follows:

Expected Strain theoretical result Result obtained E. coli − − S. sciuri− − S. pyogenes − − E. faecalis + +

This example clearly illustrates one of the advantages of the inventionby allowing greater selectivity in the characterization of themicroorganisms (E. faecalis in this application).

EXAMPLE 5 Characterization of Bacteria in Agar Medium by SimultaneousInvestigation of β-galactosidase (βGAL) and L-alanine-aminopeptidase(Ala) Activities

α-Naphthyl-β-D-galactopyranoside andL-alanyl-N,N′-dimethyl-p-phenylenediaminenaphthalenesulfonyl-ydrazideare added to a trypticase soya agar medium. This substrate is derivedfrom compound I according to the invention.

The medium is distributed and inoculated in the following way:

six dishes with pure cultures:

Escherichia coli [βGAL (+), Ala (+)]

Klebsiella pneumoniae [βGAL (+), Ala (+)]

Salmonella typhimurium [βGAL (−), Ala (+)]

Pseudomonas aeruginosa [βGAL (−), Ala (+)]

Staphylococcus xylosus [βGAL (+), Ala (−)]

Candida albicans [βGAL (−), Ala (−)].

two dishes with the mixtures of strains E. colil/S. xylosus and S.typhimurium/C. albicans.

The media are incubated at 35-37° C. After incubation of 18-24 h, onlythe dishes containing the E. coli and K. pneumoniae strains [⊖GAL (+),Ala (+)] contain blue-stained colonies.

With the mixture E. coli/S. xylosus, only the E. coli colonies are blue,those of S. xylosus remaining colorless.

Just as in Example 4, the production of the colored complex III based onindophenol thus necessitated the joint presence of two enzymaticactivities, in this case β-galactosidase and L-alanine-aminopeptidase.

However, the presence of only one or of neither of these two activitiesdoes not result in formation of the colored complex.

As is the case in Example 5, the choice of a suitable phenylenediaminederivative makes it possible to obtain a coloration which remains in theimmediate vicinity of the colonies and thus to distinguish two differentpopulations in a mixture.

This example shows that it is possible, according to the invention, tospecifically colored [sic] colonies at the surface of an agar medium andthus to characterize them. In the present case, it may be applied to thedetection of coliform bacteria which are Gram-negative bacilli havingboth β-galactosidase and L-alanine-aminopeptidase activity.

EXAMPLE 6 Combined Investigation of Three Enzymatic Activities Accordingto the Process of the Invention Application to the SimultaneousDetection of β-galactosidase (βGAL), β-glucosidase (βGLU) andL-alanine-aminopeptidase (Ala)

The reaction medium proposed in this example has the formulation:

meat peptone 10 g NaCl 5 g α-naphthyl-b-D-glucopyranoside 0.5 gL-alanyl-4-amino-2,6-dichlorophenyl- 0.5 g β-D-galactopyranoside H₂O1000 ml pH 7.0

The preparation of the medium is identical to the procedure described inExample 1.

The test for investigation of the 3 enzymatic activities is performed on5 strains cultured for 24 h at 35-37° C. on Trypticase-Soya agar. Thesestrains belong respectively to the species:

Escherichia coli [βGAL (+), βGLU (−), Ala (+)]

Klebsiella pneumoniae [βGAL (+), βGLU (+), Ala (+)]

Salmonella typhimurium [βGAL (−), βGLU(−), Ala (+)]

Stenotrophomonas maltophilia [βGAL (−), βGLU (+), Ala (+)]

Staphylococcus saprophyticus [βGAL (+), βGLU (−), Ala (+)]

The appearance of a blue coloration due to the formation of theindophenol complex is possible only if the three substrates arehydrolyzed. It thus reflects a combination of β-galactosidase,β-glucosidase and L-alanine-aminopeptidase activities in the samereaction medium, as is the case for the strain of K. pneumoniae. Theabsence of one of the activities prevents formation of the coloredcomplex.

The results obtained are as follows:

Expected Strain theoretical result Result obtained E. coli − − K.pneumoniae + + S. typhimurium − − S. maltophilia − − S. sapropriticus −−

This example once again shows the potentials of the invention forcarrying out highly selective tests.

The six examples given only describe enzymatic tests according to theinvention.

It is quite clearly possible to combine these tests with the known testsof the prior art, using chromogenic or fluorogenic substrates.

II-Identification process and device:

The principle of the medium, used with the identification process anddevice, is based on the use of the twinned substrates described above.

According to a representative example, the GRAM identification processuses a substrate based on aminobenzene or a derivative thereof, such asDiMethyl-paraPhenyleneDiamine (DMpPD). In the example which follows, thesubstrate consists of Alanine-Dimethyl-paraPhenyleneDiamine (AlaDMpPD).The activity demonstrated is thus the Alanine-aminopeptidase, thisactivity being specific for Gram-negative bacteria.

It functions as follows. Enzymatic hydrolysis causes release of theDMpPD group. Next, the DMPD binds with α-naphthol (or a derivative) byoxidative coupling facilitated by an oxidizing agent, potassiumferricyanide (K₃Fe(CN)₆). This then causes formation of a violet-graycomplex.

In a first stage, the device is defined in the following way:

a biogelytone medium containing as substrate: AlaDMpPD, and

a bottle brush impregnated with a reaction mixture consisting ofα-naphthol and potassium ferricyanide.

Another device has been defined, which is even more specific in Gramidentification and which can also be used with blood agars of Columbiatype. It consists of:

a medium consisting of a blood agar of Columbia type (with or withoutAlaDMpPD), and

a reaction mixture which impregnates a bottle brush and which isconstituted on the basis of α-naphthol, potassium ferricyanide andAlaDMpPD and then dried.

This reaction mixture contained in the bottle brush was further improvedby adding a step of drying the bottle brushes, which facilitates theirsubsequent manipulation.

The exact composition is as follows:

for the agar, presence or absence of AlaDMpPD at 0.075 gram per liter(g/l), and

for the bottle brush:

α-naphthol from 0.01 to 5 g/l, preferably from 0.1 to 1 g/l, and forexample 0.5 g/l,

potassium ferricyanide from 0.01 to 5 g/l, preferably from 0.1 to 1 g/l,and for example 0.5 g/l, and

AlaDMpPD from 0.01 to 5 g/l, preferably from 0.1 to 1 g/l, and forexample 0.35 g/l.

The results obtained in terms of sensitivity and specificity are thusthe best obtained. In this case, the detection of the reaction is notinstantaneous. It takes 15 to 30 seconds (s) to observe the appearanceof any coloration, this being the time required for hydrolysis of thesubstrate by the bacterial aminopeptidase.

Sixty-one strains on different media such as: Polyvitex chocolate agar(registered trademark), CPS ID2 medium (registered trademark), TSA +/−blood and Columbia +/− blood were tested with bottle brushes impregnatedwith α-naphthol, potassium ferricyanide and AlaDMpPD and then dried.This study showed that this system was compatible with the majority ofconventional media and common chromogenic agents.

To further improve the reaction time, a reaction activator isincorporated into the medium.

In this context of activating the reaction, it was found that theaddition to the medium of a small amount of AlaDMpPD and/or ofα-naphthol allows the reaction to be detected without reducing thesensitivity. Furthermore, the detection time is thus considerablyreduced, since it is taken from 0 to 10 s [sic]. This small amountcorresponds to a concentration of from 0.01 to 0.5 g/l, preferably from0.05 to 0.1 g/l, and for example 0.075 g/l.

According to one embodiment, it is also possible to add a binder to thebottle brush. The addition of a binder makes it possible both to gathertogether the fibers in the head of the bottle brush and to limit therelease of products present on the bottle brush into the inoculumsuspension. Such a binder can consist of PolyVinylPyrrolidone (PVP),which in fact makes it possible not only to satisfy the above criteriabut also to substantially increase the specificity. In the compositiondescribed above with the concentrations, the PVP is introduced into theconstituents of the viscose head of the bottle brush in a concentrationof from 1 to 50 g/l, preferably from 10 to 25 g/l, and for example 15g/l.

1. Experiment

An evaluation of this medium was thus carried out on one hundred andthirty-three (133) strains divided among the following species. Table 1below lists the species tested and the number of strains for eachspecies.

TABLE 1 Species tested and number of strains for each species Number ofSpecies strains Acinetobacter baumanii 2 Acinetobacter junii 2Citrobacter freundii 3 Citrobacter spp 1 Enterobacter cloacae 3Enterobacter intermidis 1 Enterobacter spp 1 Escherichia coli 4Klebsiella oxytoca 5 Klebsiella pneumoniae 4 Morganella morganii 4Proteus mirabilis 5 Proteus vulgaris 5 Providencia stuartii 4Pseudomonas fluorescens 5 Serratia marcescens 4 Bacillus cereus 4Bacillus lentus 1 Bacillus mycoides 2 Bacillus subtilis 2Corynebacterium aquaticum 2 Corynebacterium ulcerans 2 Enterococcusfaecalis 5 Enterococcus faecium 3 Enterococcus gallinarum 3 Haemophilushaemolyticus 2 Haemophilus influenzae 2 Lactobacillus casei 4 Listeriamonocytogenes 4 Staphylococcus aureus 3 Staphylococcus epiderinidis 3Staphylococcus haemolyticus 3 Staphylococcus saprophyticus 3Streptococcus agalactiae 4 Streptococcus pneumoniae 3 Streptococcuspyogenes 3 Candida albicans 3 Candida glabrata 3 Candida guilliermondii1 Candida kefyr 1 Candida krusei 3 Candida parapsilosis 1 Candidatropicalis 4 Trichosporon asteroides 1 Trichosporon mucoides 1 TOTAL133 

Table 2 lists the results obtained:

TABLE 2 Sensitivity and specificity of the species tested Sensitivity93% Specificity towards Gram + bacteria 81 to 90% Specificity towardsyeasts  89 to 100%

Among the results obtained on the strains tested, certain strains arefalse negatives. These false negatives are two (2) of the four (4)strains of P. aeruginosa, two (2) of the five (5) strains of P.fluorescens and one (1) of the two (2) strains of H. influenzae. Certainstrains are false positives. These false positives consist of the two(2) strains of C. ulcerans, one (1) of the five (5) strains of E.faecalis, the three (3) strains of S. pyogenes, two (2) of the four (4)strains of S. agalactiae, the strain of C. guilliermondii and the strainof C. parapsilosis.

2. Readability of the test

Virtually all of the Gram-negative bacteria, except for the Pseudomonasbacteria, give a sharp violet-gray coloration whose intensity isstrictly greater than 2 (on a semiquantitative scale up to 4), while theGram-positive bacteria or those which are false-positives, except for S.pyogenes, have a coloration intensity of less than 1 on this same scale.

3. Detection time

About 90% of the Gram-negative bacteria are detected between 0 and 10 s;most of the Gram-positive bacteria or those which are false-positivesare detected after 10 s, apart from the strains of S. pyogenes.

The current medium thus has good sensitivity and good specificity. Theother false-positive strains are generally of very weak intensity andhave a detection time of greater than 10 s. These strains are thusrather [sic] dubious strains rather than false-positives.

4. Definition of the bottle brush

The bottle brush model, which is particularly advantageous, is a bottlebrush which satisfies the following criteria:

a plastic stick 2.5 millimeters (mm) in diameter and 150 mm in length,and

with a viscose head less than or equal to 4.5 mm in diameter.

5. Method for manufacturing and manipulating the bottle brushes

The manufacturing conditions are as follows:

soaking in a solution as defined above for 2 to 3 minutes (min),

drying for 2 hours (h) at 37° C., and

packaging in a leakproof plastic bag protected from the light.

6. Packaging of the bottle brushes

The bottle brushes can be stored, for example, in a sterile manner(gamma rays) in wrapping containing one (1), twenty five (25) or onehundred (100) to one thousand (1000) of these bottle brushes. However, apackaging of from twenty (20) to twenty five (25) bottle brushes notnecessarily sterilized after drying is entirely conceivable. Thepackaging is carried out with or without a desiccant, and sheltered fromthe light.

7. Critical parameters

The use of the twinned substrates is thus identical to that outlined atthe start of the description. Thus, the head of the bottle brushcontains at least two molecules. Thus, in the example given, a firstmolecule (AlaDMpPD) consists of a nonchromogenic labeling portion(DMpPD) associated with at least one specific target portion (alanine)of the enzyme (Alanine-aminopeptidase) and a second molecule consistingof another nonchromogenic substance (α-naphthol), the nonchromogeniclabeling portion (AlaDMpPD), once released, reacts with the secondmolecule (α-naphthol) to form a chromogenic molecule.

The use of the bottle brush serves as a support for reagents and allowsthe preparation of the inoculum suspension, which will be used in otheridentification and antibiotic assay systems, on the basis of the samestrains which were evaluated above. This thus considerably reduces theerrors during inoculation of new “similar” microorganisms for carryingout the following identification and antibiotic assay operations, etc.

Furthermore, it is also possible to prepare bottle brushes for othertypes of tests, for example oxidase, indole, esterase, phosphatase, etc.

What is claimed is:
 1. A chromogenic substrate for detecting thepresence of enzymatic activity of at least two enzymes, characterized inthat said substrate consists of at least two molecules, a first moleculeconsisting of a nonchromogenic labeling portion associated with at leastone specific target portion of the first enzyme and a second moleculeconsisting of another nonchromogenic labeling portion associated with aspecific target portion of the second enzyme, and in that thenonchromogenic labeling portions, once released, react to form achromogenic molecule.
 2. The substrate, according to claim 1,characterized in that the nonchromogenic labeling portion of the firstmolecule consists of aminobenzene or a derivative thereof:

in that the nonchromogenic labeling portion of the second moleculeconsists of α-naphthol or a derivative thereof:

and in that the chromogenic molecule obtained consists of:


3. The substrate, according to claim 2, characterized in that theradical R₁ consists of —OH, —SH or

in that the radical R₁₀ consists of —H or an atom, such as —Br, —Cl or—I, or a group oE atoms, such as —SH, which can be removed during theoxidative coupling, and in that each radical R₂ to R₉, R₁₁, or R₁₂consists of —H, —OH, —Br, —Cl —I or other more complex substituents,such as —CH₃, —CH₂CH₃, —OCH₃, —OCH₂CH₃ or —COOH.
 4. The substrate in thecase in which the radical R₁ consists of

according to claim 2, characterized in that X and/or Z consists of —H,or other more complex substituents, —CH₃, —CH₂CH₃,


5. The substrate, according to claim 2, characterized in that at leastone of the pairs of radicals R₂/R₃ and R₄/R₅ consists of an aromatic,alicyclic or heterocyclic system.
 6. Process for detecting at least twoenzymatic activities via substrates, charcterized in that said processconsists in: placing molecules consisting of at least two substrates inthe presence of at least one type of bacteria microorganisms which iscontained in a test sample, waiting for the bacteria to Hydrolyze thesubstrates, and detecting the enzymatic activities based on theformation of a colore complex from the oxidative coupling of the twolabeling portions.
 7. Process, according to claim 6, characterized inthat the molecules are present in an absorbent material and are placedin contact with the sample, and after detection, the microorganismswithdrawn are resuspended in order to allow subsequent analyses,identifications, or antibiotic assays.
 8. Process according to claim 6,characterized in that the molecules and other compounds forming part ofthe reaction composition, optionally contained in the absorbentmaterial, are: the first molecule consisting of aminobenzene or aderivative thereof, and/or the second molecule consisting of α-naphtholor a derivative thereof, optionally combined with a potassiumferricyanide oxidixing agent.
 9. Process, according to claim 8,characterized in that it comprises a reaction activator, a small amountof the first molecule and/or of the second molecule, which is present inthe sample to be tested.
 10. Process, according to claim 8,characterized in that the composition comprises a PolyVinylPyrrolidone(PVP) binder.
 11. Process, according to claim 8 for the Gramidentification of a bacterial species to be tested, characterized inthat the first molecule consists of AlaDMpPD, and in that the secondmolecule consists of α-naphthol.
 12. Process, according to claim 8,characterized in that the composition of the absorbent material is asfollows: α-naphthol from 0.01 g/1 to 5 g/l, potassium ferricyanid from0.01 g/l to 5 g/l, and AlaDMpPd from 0.01 g/l to 5 g/l, from. 13.Process, according to claim 9, characterized in that the activatorconsists of Ala-DMpPD at a concentration of from 0.001 g/l to 0.5 g/l.14. Process, according to claim 10, characterized in that thecomposition also comprises from 1 g/l to 50 g/l of PVP.
 15. A substrate,according to claim 1, for detecting an enzymatic activity of peptidase.16. A device for detecting, in a sample, at least two enzymaticactivities, characterized in that said device comprises a support inwhich an absorbent viscose material is placed at least one substrateaccording to claim 1, and the sample, wherein said support made ofplastic, being inert with respect to the absorbent material, to thesubstrate(s) and to the sample.